1. Field of the Invention
The present invention relates to biological homogeneous assays. In particular, the invention relates to a corrected method for biological homogeneous assays.
2. Description of Related Art
Ullman et al. first applied luminescence resonance energy transfer (LRET) technology to produce a homogeneous immunoassay using fluorescein and rhodamine as donor and acceptor, respectively. The use of conventional organic fluorophore pairs for homogeneous LRET applications proved not to be practical because the lifetimes of the donor and acceptor were too short, translating to background levels that were too high to make the technology practical. Ideally, a donor luminophore should combine high brightness, high stability and a long luminescence lifetime to be practical for high sensitivity diagnostic applications. The longer luminescent lifetimes of lanthanide donors make them appealing for LRET applications because time-gating techniques can remove the background levels that hamper conventional LRET technologies.
Variations in sample properties including color, viscosity, pH, presence/absence of solids, etc. have the potential to impact assay results. With a heterogeneous assay, this is not usually a concern when the sample result is read by absorbance or luminescence because the sample is washed from the reaction site before the sample is read. With a homogeneous assay, the differences in sample properties can affect the luminescence values. Samples with the same amount of analyte can and do yield different raw luminescent data depending on the properties of the sample. Interfering constituents in samples can absorb the excitation light, donor emission, absorbance emission more or less than a control sample, thereby affecting sample results.
Luminescent lanthanide compounds are known to have long lifetimes (on the order of milliseconds), while conventional organic dyes have lifetimes in the range of nanoseconds. This unique property enables the time gated removal of short-lived background interference that hampers conventional luminescence techniques. As shown in Prior Art FIG. 1, an excitation flash is delivered to a complex biological matrix followed by a short delay (for example 100 μs) to allow unwanted interfering background noise to dissipate. Then data is collected during an integration window that is much longer than the undesirable background (for example 400 μsec) yielding a signal almost free of interference.
Lanthanide dyes can act as donors with conventional organic dye acceptors. When the donor and acceptor are brought into close proximity, excitation energy absorbed by the donor is transferred to the acceptor by a Förster mediated dipole-dipole coupling. The emitted light from the acceptor can be measured in a time-resolved mode, yielding low-noise LRET data compared to data obtained from conventional LRET pairs.
The improved data quality is readily apparent in Prior Art FIG. 2 when comparing the emission of fluorescein (conventional settings) with the emission of an exemplary lanthanide dye, LUMI4-TB (a terbium chelate trademarked by Lumiphore, Inc.) using time-resolved settings. LUMI4-TB is a member of a new class of luminescent hydroxyisophthalamide chelates that combines a high quantum yield (60%) and absorption coefficient and exhibits high stability in aqueous environments. The properties and structure of luminescent hydroxyisophthalamide chelates of lanthanides are discussed in U.S. patent application Ser. No. 12/521,919, “Multi-Color Time Resolved Fluorophores Based on Macrocyclic Lanthanide Complexes,” Butlin, Corneillie, and Xu; filed Jan. 25, 2008.
Prior Art FIG. 2 demonstrates the reduced background and improved linear range achieved with time-resolved luminescence. In this experiment, fluorescein exhibits a linear range down to ˜1.0 nM (conventional settings). Time-resolved measurements of LUMI4-TB exhibit a much larger linear range (down to below 10 pM), which is achieved by gating out short-lived interference attributable to the lamp source, optics, matrix components, etc.
Prior Art FIG. 3 shows the absorption and emission spectra for an exemplary lanthanide dye, LUMI4-TB. Broad absorption of the sensitizing 2-hydroxyisophthalamide chelating unit is centered at 340 nm. The emission spectrum is characteristic of luminescent terbium complexes with the dominant peaks centered at 545 nm and 490 nm. The large separation between absorption and emission peaks (Stokes Shift) eliminates the reabsorption of emitted luminescence that hampers the performance of conventional organic dyes.
A schematic representation of an exemplary competitive, homogeneous assay technology is shown in Prior Art FIGS. 4A, 4B, 4C, and 4D. As shown in FIG. 4A, an antibody 405 is coupled to an acceptor luminophore 410 (e.g., fluorescein) to serve as an acceptor reagent with binding sites 415 that are specific for analyte 420 and the competitor conjugate donor 425 that is derived from the analyte component and a luminophore (e.g., LUMI4-TB). As shown in FIG. 4A, the concentration of the analyte 420 is low relative to the concentration of the competitor conjugate donor 425.
As shown in FIG. 4B, excitation energy is first absorbed by the competitor conjugate donor 425. When the donor and acceptor are brought into close proximity (e.g., through binding to the antibody 405), energy is transferred from the competitor conjugate donor 425 to the acceptor 410 by a Förster mediated dipole-dipole coupling. The acceptor 410 subsequently produces a LRET signal at the wavelength of the acceptor luminophore 410. In the presence of a low amount of analyte the LRET signal is near its maximum, as the competitor conjugate donor 425 binds unimpeded to the analyte-specific antibody 405.
As shown in FIG. 4C and FIG. 4D, the concentration of the analyte 420 is high relative to the concentration of the competitor conjugate donor 425, and the LRET signal is low, since the analyte 420 out competes the competitor conjugate donor 425 that carries the lanthanide donor.
For laboratory tests, homogeneous assay methods are preferable to heterogeneous methods because test procedures are simple and fast, requiring no washing steps. LRET is a homogeneous method that does not require enzyme inactivation or reformation to generate a signal and avoids some of the limitations of other homogeneous assay technologies. This assay platform relies on LRET between complementary molecules labeled with either a lanthanide donor or an acceptor (e.g., fluorescein). The key component to a practical LRET assay is the donor luminophore, which ideally should combine high brightness, high stability and low background to attain the sensitivity required for detecting drugs of abuse in oral fluid. The longer lifetimes of lanthanide donors make them appealing for LRET applications because time-gating techniques remove the background levels that hamper conventional LRET technologies. LUMI4-TB incorporates four isophthalamide chelating units and achieves an unparalleled level of brightness when coordinated to terbium. The luminescent lanthanide technology has been combined with high affinity and high specificity antibodies for drugs to develop this oral fluid drug assay technology.
Oral fluid has become an important matrix for testing because samples are relatively easy to collect, are more reflective of recent use and more closely tracking blood levels, the reference standard. Testing for drugs in oral fluid presents a challenge for current homogeneous laboratory testing technologies and point-of-care testing (POCT) products, which were developed for testing in urine. While detection levels for laboratory and lateral flow POCT assays for urine are in the 50-2000 ng/mL range for drugs, the levels in oral fluid are sometimes an order of magnitude or more lower. More sensitive technologies are needed.
In order to correct for the variability between samples that can occur in a homogeneous assay, techniques involving additional measurements may be used. For example, U.S. Pat. No. 6,861,264, “Method of Measuring the Luminescence Emitted in a Luminescent Assay,” Mabile et al, issued Mar. 1, 2005, teaches the use of measurements taken at two difference wavelengths emitted by a single sample volume.
Thus there is a need for a rapid, homogeneous assay method for measuring parent drug compounds in oral fluid. There is also a need for a method of measurement correction that is applicable at a single wavelength.